Basic helix-loop helix (bHLH) transcription factors function as important regulators of differentiation processes in many tissues. These proteins act as dimers with a tissue specific family member interacting with a ubiquitously expressed family member. The dimeric proteins, but not the monomeric subunits, form a composite DNA-binding domain with their basic sequences and can interact with a DNA response element, the E-box. Although no epithelial-specific bHLH proteins have been identified to date, our preliminary experiments suggest that such factors exist and play a role in coupling proliferation and differentiation in keratinocytes. Regulation of the activities of bHLH proteins is achieved through multiple mechanism. During differentiation, the activities of bHLH proteins are modulated by expression of naturally occurring dominant negatives, the Id proteins (inhibitor of DNA binding; inhibitor of differentiation). Id proteins effectively sequester bHLH proteins leading to a decrease in the amounts of intracellularly available bHLH proteins. Our preliminary experiments have shown that Id proteins are expressed in primary cultures of human foreskin keratinocytes. The expression of some of the family members is regulated during differentiation. Most strikingly, Id1 expression is strongly induced during differentiation. Id-expressing keratinocyte populations have an increased life span and immortalized cell lines overexpressing Id1 have been obtained. Id1 immortalized cells have a differentiated morphology. Moreover, the function of the p53 growth regulatory pathway is abrogated in these cells. This strongly suggests that Id proteins, in particular Id1, can uncouple cellular proliferation in differentiating keratinocytes. We propose the following specific aims: (1) To measure the transcriptional activities of bHLH proteins during keratinocyte differentiation, (2) to analyze the effect of the Id1 protein on keratinocyte differentiation, and (3) to define the mechanism of p53 inactivation in Id1 immortalized keratinocytes. These studies will provide for the basis to eventually identify the molecular targets of Id proteins in keratinocytes.